Comparison of the virulence of exopolysaccharide-producing Prevotella intermedia to exopolysaccharide non-producing periodontopathic organisms.

BACKGROUND: Evidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built. METHODS: This study evaluates the role of EPS in Prevotella intermedia for the expression of virulence. This evaluation was accomplished by comparing EPS-producing P. intermedia strains 17 and OD1-16 with non-producing P. intermedia ATCC 25611 and Porphyromonas gingivalis strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis. RESULTS: EPS-producing P. intermedia strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 107 colony-forming units (CFU). In comparison, P. intermedia ATCC 25611 and P. gingivalis ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (109 CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, P. intermedia strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes. CONCLUSIONS: These results demonstrate that the production of EPS by P. intermedia strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the host's innate defence response.

Biofilm-like structures and pathogenicity of Escherichia hermannii YS-11, a clinical isolate from a persistent apical periodontitis lesion.

Escherichia hermannii, formerly classified as enteric group 11 of Escherichia coli, is considered to be nonpathogenic. In this report, we described some of the pathogenic properties of a viscous material-producing E. hermannii strain YS-11, which was clinically isolated from a persistent apical periodontitis lesion. YS-11 possessed cell surface-associated meshwork-like structures that are found in some biofilm-forming bacteria and its viscous materials contained mannose-rich exopolysaccharides. To further examine the biological effect of the extracellular viscous materials and the meshwork structures, we constructed a number of mutants using transposon mutagenesis. Strain 455, which has a transposon inserted into wzt, a gene that encodes an ATP-binding cassette transporter, lacked the expression of the cell surface-associated meshwork structures and the ability to produce extracellular materials. Complementation of the disrupted wzt in strain 455 with an intact wzt resulted in the restoration of these phenotypes. We also compared these strains in terms of their ability to induce abscess formation in mice as an indication of their pathogenicity. Strains with meshwork-like structures induced greater abscesses than those induced by strains that lacked such structures. These results suggest that the ability to produce mannose-rich exopolysaccharides and to form meshwork-like structures on E. hermannii might contribute to its pathogenicity.

Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17.

BACKGROUND: Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays. RESULTS: Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (sigmaE; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media. CONCLUSION: These results demonstrate that the capacity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.

A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPalpha) conjugative machineries and their cognate Escherichia coli host strains.

We describe the construction of the pSW family of conditionally replicating plasmids which are based on the IncX oriV origin (oriV(R6Kgamma)) of replication that is dependent on the pir-encoded protein. We constructed several Escherichia coli derivatives expressing pir from different chromosomal loci, and the pir gene could be transduced by phage P1 to any E. coli strain. These chromosomal constructions generate dapA and thyA knockouts, which lead to diaminopimelate or thymidine auxotrophies, respectively, and they serve to provide absolute counterselection even in rich media. These strains can be easily counterselected if used in plasmid transfer experiments into markerless recipients, and they have been demonstrated to work efficiently in E. coli xVibrio or E. coli xBartonella matings. We constructed different pSW plasmids carrying either the oriT(RP4) or the oriT(R388), and we demonstrated that these derivatives can be efficiently transferred using RP4 and R388 conjugation machineries, respectively. We also constructed two plasmids expressing the R388 conjugation machinery, but lacking the oriT(R388). We demonstrated that these plasmids enabled efficient and exclusive transfer of a pSW-oriT(R388) derivative from E. coli to V. cholerae, and we offer an alternative to the popular RP4-based delivery system.

A new family of conditional replicating plasmids and their cognate Escherichia coli host strains.

We constructed a family of conditionally replicating plasmids, the pTX1 family, which are based on the IncPalpha oriV origin of replication that is dependent on the trfA-encoded protein. We constructed several Escherichia coli derivatives expressing trfA from different chromosomal loci, which can be transduced by phage P1 to any E. coli strain. The pTX1 plasmids also carry the oriTRP4 origin of transfer, and can be conjugated to E. coli, Vibrio cholerae and likely to a broad range of bacteria from the commonly used donor strains SM10 and S17-1, which sustain replication of the plasmids through the trfA gene carried by their integrated RP4. If TrfA is not provided in trans, these plasmids behave as suicide vectors. As such they can be used as a platform for a variety of applications such as those developed on the popular conditionally replicating plasmids carrying the oriVR6Kgamma origin of replication that is controlled by the Pi protein. Their ability to be used as efficient suicide vectors for gene disruption in V. cholerae has been demonstrated.